The first component of complement (C1) occurs in serum as an inactive macro-molecular complex consisting of three subunits: C1q, C1r and C1s. Binding of C1 to antigen-antibody aggrgates activates the C1s subunit, which through limited proteolysis of components C4 and C2, initiates a sequence of reactions resulting in immune hemolysis. A considerable body of evidence has accumulated indicating that C1s exists as a zymogen in the native C1 complex and is susceptible to direct proteolytic activation. Proper evaluation of proposed internal and autocatalytic activation mechanisms requires characterization of the covalent changes attending activation of the first component of complement. Methods currently used to prepare zymogens of other serine proteases will be adapted to purification of C1s on a preparative scale. During activation the purified zymogen will be monitored for changes in N-terminal sequence, electrophoretic mobility, esterase activity, base consumption at constant pH and spectral parameters. Evidence for enzymatic activity intrinsic to the zymogen will be sought using substrates and active-site reagents. Systematic structural and enzymological studies will also be initiated on the active enzymatic subunit, C1s. These studies will be directed toward understanding the restricted specificity of C1s as well as the mechanisms by which this specificity is modulated. Such a program would involve isolation of active-site peptides, sequenator studies of intact and autolyzed preparations, examination of the susceptibility of peptide substrates to hydrolysis by C1s and investigation of the effects of selected active-site titrants.